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Extrachromosomal gene expression vectors that contain native genomic gene expression elements have numerous advantages over traditional integrating mini-gene vectors. In this protocol chapter we describe our work using episomal vectors where expression of a cDNA is controlled by a 10 kB piece of genomic DNA encompassing the promoter of the low density lipoprotein receptor. We explain methods to sub-clone large genomic inserts into gene expression vectors. We also illustrate various methods employed to ascertain whether expression from these vectors is robust and physiologically relevant by investigating their sensitivity to changes in cellular milieu. Delivery of gene expression vectors in vivo is also described using hydrodynamic tail vein injection, a high pressure, high volume tail vein injection used for liver-directed gene transfer.

Original publication

DOI

10.1007/978-1-61779-099-7_2

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2011

Volume

738

Pages

19 - 40

Keywords

Animals, CHO Cells, Chromosomes, Artificial, Bacterial, Cricetinae, Cricetulus, DNA, Gene Expression, Genes, Reporter, Genetic Engineering, Genetic Therapy, Genetic Vectors, Genome, Humans, Hyperlipoproteinemia Type II, Lipoproteins, LDL, Liver, Luciferases, Mice, Molecular Imaging, Organ Specificity, Plasmids, Promoter Regions, Genetic, Protein Transport, Receptors, LDL, Transfection, Transgenes